NMR sample preparation of membrane

proteins in phospholipid bilayers

Sang Ho Park

University of California, San Diego

Structure determination of membrane proteins by solid-state NMR spectroscopy.

Step 1 Prepare a sample of uniformly 13C/15N labeled

proteoliposomes

Step 2

Step 3

Step 4

Resolve individual signals with MAS solid-state

NMR experiments

Assign each signal to

a specific residue

Measure orientation-dependent

frequencies for each residue

Step 5 Calculate three-dimensional

structure

Marassi et al. Methods (2011)

Highly stable samples are required to perform structural studies on membrane proteins by solid-state NMR spectroscopy.

General procedure from bacterial expression to

reconstitution of membrane proteins into lipid bilayers.

By using an efficient protocol, it takes 3 days from the bacterial culture to the final NMR sample of membrane proteins: day 1,

growth of E. coli culture and preparation of IBs; day 2, purification and refolding; day 3, preparation of CXCR1 proteoliposomes

and the final NMR sample. Every step needs optimization for each membrane proteins.

Park et al. BBA (2012)

Efficient expression can be achieved using E. Coli codon

optimized genes.

• Genes are clustered by using factorial correspondence analysis into three classes. Class I contains genes involved in most

metabolic processes. Class II genes correspond to genes highly and continuously expressed during exponential growth. Class

III genes are implicated in horizontal transfer of DNA. One can see that the distribution of codons in class III genes is more or

less even, whereas it is extremely biased in class II genes (in particular, codons terminated in A are selected against).

• Replacing codons that are rarely found in highly expressed E. coli genes with more favourable codons throughout the whole

gene. Codons that have been associated with translation problems in E. coli are marked in red box.

Expression optimized host strains are used for

overexpression of target proteins

• C41(DE3) and C43(DE3) cells for toxic protein expression

– Dumon-Seignovert et al. Protein Expr Purif (2004)

• Non-DE3 BL21 cells for expression with mild lac or tac promoter

• Rare codon optimized host cells

– BL21 (DE3) CodonPlus-RIPL

– BL21 (DE3) CodonPlus-RIL

– BL21 (DE3) CodonPlus-RP

– Rosetta or Rosetta (DE3)

– BL21 CodonPlus-RIL

– BL21 CodonPlus-RP

If the E coli codon optimized genes are not available, rare codon optimized host cells can be used for expression of rare codon

containing genes.

High level expression of fusion protein

Common fusion partners for bacterial expression of membrane proteins• KSI: Ketosteroid Isomerase

• GST: Glutathione S-transferase

• TrpΔLE: Trp-LE leader sequence

• Bcl-XL: B-cell lymphoma-extra large

• MBP: Maltose Binding Protein

pGEX2a-CXCR1-His

6119 bp

APr

GST

CXCR1 Rezeptor

lac I

His tag

Ptac

ORI

rrnBT1T2

Thrombin cleavage site

CXCR1 receptor

TrpΔLE

p7

BL21(DE3) BL21

p7 CXCR1

No single solution exists for successful production of all recombinant membrane proteins. Instead, it is beneficial to have

access to a wide range of expression tools.

Purification of Vpu from HIV-1

1. Supernatant after cell lysis

2. Inclusion bodies

3. Ni-NTA purified fusion protein

4. CNBr cleaved protein

5. HPLC purified protein

SDS-PAGE HPLC

Fusion partner

Fusion partner

Acquiring high-quality NMR spectra demands the production of samples with high levels of purity and excellent homogeneity

throughout the sample.

Size exclusion chromatography of human chemokine

receptor CXCR1

Pure monomeric receptor

The CXCR1 samples purified by nickel affinity chromatography that contain a small amount of higher oligomers must be

immediately loaded onto the size exclusion chromatography, since trace amount of oligomers (and/or other impurities)

apparently facilitate the formation of larger amounts of higher oligomers.

Reconstitution of CXCR1

1) Mixed micelle solutions were prepared by dissolving the DMPC

powder with 0.5% SDS in 20 mM HEPES buffer pH 7.3, 50 mM

NaCl with a final lipid concentration of 10 mg/ml.

2) Purified monomeric CXCR1 was added to the mixed micelle

solution at a protein-to-lipid ratio of 1:10 or 1:5 (w/w).

3) Incubated for 1 h at room temperature.

4) Detergents were removed to obtain highly pure proteoliposomes.

Detergent removal

• Adsorption to BioBeads.

– fast and effective for low-CMC detergents

– lipid loss due to non-specific adsorption

• Complexation to methyl-β-cyclodextrin.

– Applicable to broad range of detergents

– Reproducible without loss of lipids

• Co-precipitation with potassium chloride.

– Only Suitable for detergents having sulfate head groups (e.g. SDS)

– Reproducible without loss of lipids

Lipid analysis using HPLC-ELSD system

Before reconstitution After reconstitution

retention (ml)

HPLC with Evaporative Light Scattering Detector (ELSD)

It is important for sample quality control to make sure that there are no detergents left after reconstitution in order to obtain

highly pure proteoliposomes for NMR experiments.

Sample preparation for MAS NMR experiments

1) Concentrate proteoliposomes using ultracentrifugation.– 145,000 g, 2 hr – overnight, 15oC.

– Spinning time needs optimization for RA solid-state NMR experiments.

2) Transfer proteoliposomes to MAS rotor using Microman® (Gilson).– Concentrated proteoliposomes are highly viscous.

– Microman® M50 and M10 are used for 3.2 mm rotor and 1.3 mm rotor, respectively.

– Pack the sample evenly by using a centrifuge to ensure that the rotor is properly balanced during MAS.

1) Place an insert and seal the rotor with a rotor cap.– An insert improves shimming and RF in homogeneity.

– Check the integrity of the cap before use and make sure it fits snugly on the rotor.

Rotors and tools for MAS sample preparations

3.2 mm rotor up to 24 kHz spinning rate

for moderate spinning MAS experiments

1.3 mm rotor up to 67 kHz spinning rate

for fast spinning MAS experiments

Microman® M50 and CP50

for 3.2 mm rotor packing

Microman® M10 and CP10

for 1.3 mm rotor packing

http://www.theresonance.com/2014/categories/material-science/nmr-tutorial-mas-rotor-filling

https://www.youtube.com/watch?v=K_0yPPmFYdE#t=118